We recommend using Alt-R A.s. Cas12a (Cpf1) Nuclease V3 combined with the Alt-R CRISPR-Cas12a (Cpf1) crRNA to generate a ribonucleoprotein (RNP) editing complex. The Alt-R Cas12a (Cpf1) Electroporation Enhancer is critical for optimal delivery by electroporation
and is recommended for all experiments using this transfection method. View the Alt-R CRISPR-Cas12a (Cpf1) System User Guide for guidance on electroporation and delivery of the RNP into your cell lines.

CRISPR-Cas12a (Cpf1) crRNA

Customer-defined crRNA that will bind to 21–24 bases opposite to the TTTV, PAM sequence. Prices shown are for each crRNA. Plates require a minimum order of 24 crRNAs.

Unlike S. pyogenes Cas9, which cleaves most potential NGG PAM sites to some degree, some of the tested TTTV
sites show no cleavage by A.s. Cas12a nuclease. We recommend using positive control crRNAs (see below for suggested
sequences) to establish that your cells can be edited by Cas12a1. In addition, we recommend testing 3 or more crRNAs
per target gene.

Custom CRISPR solutions

Don’t see what you’re looking for? We are continually expanding our CRISPR product line, and we may have what you need. If
you are interested in custom libraries, other CRISPR enzymes, formulations, or other CRISPR tools contact our CRISPR
experts today to discuss customized solutions for your research: CRISPR@idtdna.com.

Alt-R CRISPR-Cas12a System

CRISPR-Cas12a genome editing method uses the Cas12a endonuclease to generate double-stranded breaks that contain a staggered 5′ overhang. Cas12a only requires a single CRISPR RNA (crRNA) to specify the DNA target sequence (Figure 1). After cleavage, DNA is then repaired by non-homologous end-joining (NHEJ) or homology-directed recombination (HDR), resulting in a modified sequence. Alt-R CRISPR-Cas12a reagents provide essential, optimized tools needed to use this pathway for genome editing research. A brief comparison of CRISPR-Cas9 and CRISPR-Cas12a is provided at the end of this section.

Cas12a-specific carrier DNA required for efficient electroporation of the RNP

Related reagents and kits

Cas12a positive controls

Order as custom crRNAs or oligos

HPRT crRNAs (human, mouse, or rat): To show that Cas12a editing is occurring in your system during experimental optimization or troubleshooting

HPRT PCR primers (human, mouse, or rat): To detect genome editing in experiments with HPRT crRNAs; for use with the Alt-R Genome Editing Detection
Kit

Alt-R Genome Editing Detection Kit

For mutation detection and estimating editing efficiency

Components for genome editing

Cas12a endonuclease from Acidaminococcus sp. BV3L6 along with a crRNA is capable of mediating genome editing in mammalian cells (Figure 2). The Alt-R CRISPR-Cas12a System includes 3 main components: an optimized crRNA, A.s. Cas12a endonuclease, and an electroporation enhancer. While electroporation of Cas12a endonuclease as part of an RNP is the preferred method, the Alt-R CRISPR-Cas12a crRNA is also compatible with A.s. Cas12a from any source, including cells that stably express A.s. Cas12a endonuclease.

Figure 2. Components of the Alt-R CRISPR-Cas12a System for directing Cas12a endonuclease to genomic targets. Alt-R Cas12a (Cpf1) crRNA forms a complex with Cas12a endonuclease to guide targeted cleavage of genomic DNA. The cleavage site is specified by the protospacer element of the crRNA (thick green bar). The crRNA protospacer element recognizes 21 nt on the opposite strand of the TTTV PAM site. The PAM site must be present immediately upstream of the protospacer element for cleavage to occur. PAM = protospacer adjacent motif; V = A, C, or G

Alt-R CRISPR-Cas12a (Cpf1) crRNA and design tips

The Alt-R CRISPR-Cas12a (Cpf1) crRNA is a single, 40–44 base, guide RNA, comprised of a 20 base constant region (loop domain) and
a 20–24 base target-specific region (protospacer domain). We typically recommend a 21 base protospacer domain for optimal
activity. All Alt-R CRISPR-Cas12a (Cpf1) crRNAs are synthesized with proprietary chemical modifications, which protect the crRNA
from degradation by cellular RNases and further improve on-target editing performance. For crRNAs used with A.s. Cas12a,
identify locations in your target region with the protospacer adjacent motif (PAM) sequence, TTTV, where V is A, C, or
G. Your Alt-R CRISPR-Cas12a (Cpf1) crRNA will bind to the DNA strand opposite to the PAM sequence (Figure 2). Do not include the
PAM sequence in your crRNA design. An example of a correct crRNA sequence is shown in Figure 3.

After you enter your 20–24 base target sequence, 20 additional bases and the necessary modifications will automatically be
added by the order entry system for a total of 40–44 RNA bases. These additional bases and modifications are necessary
to create a complete Alt-R CRISPR-Cas12a (Cpf1) crRNA. The system will also convert the final sequence to RNA—enter DNA bases
only into the ordering tool (Figure 3).

Figure 3. How to enter your Cas12a (Cpf1) crRNA target sequence. Because the crRNA recognizes and binds 21 bases on the DNA strand opposite from the TTTV sequence of the PAM site, order your crRNA by entering the 20–24 bases downstream of the PAM site, in the forward orientation as shown. Enter only DNA bases into the order entry tool. If you are pasting your CRISPR target site from an online design tool, make sure you verify the correct strand orientation. Do not include the PAM site in your design. Common incorrect design examples are shown in red. PAM = protospacer adjacent motif; V = A, C, or G

In contrast to Streptococcus pyogenes Cas9, which recognizes an NGG PAM sequence, the A.s. Cas12a PAM sequence is TTTV, which permits targeting of DNA sequences in AT-rich regions of the genome.

Alt-R Cas12a (Cpf1) Electroporation Enhancer

The Alt-R Cas12a (Cpf1) Electroporation Enhancer is a Cas12a-specific carrier DNA that is optimized to work with the Amaxa® Nucleofector®
device (Lonza) and the Neon® Transfection System (Thermo Fisher) for increased transfection efficiency and therefore,
increased genome editing efficiency.Alt-R CRISPR-Cas9 Control crRNAs and PCR Assays

Positive controls

crRNAs

Positive control crRNAs can be used to show that Cas12a editing is occurring in your experiments, which can be useful when
you are optimizing RNP delivery conditions or if you need to troubleshoot your experiments.

Attention: Unlike S. pyogenes Cas9, which cleaves most potential NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cas12a nuclease. We recommend using positive control crRNAs to establish that your cells can be edited by Cas12a. In addition, we suggest testing 3 or more crRNAs per target gene.

Our scientists have designed and tested positive control crRNAs targeting HPRT (Figure 4). To order, copy and paste the appropriate
sequence in the Cpf1 crRNA ordering page:

Human HPRT1, Cas12a Positive Control crRNA: GGTTAAAGATGGTTAAATGAT

Mouse Hprt, Cas12a Positive Control crRNA: GGATGTTAAGAGTCCCTATCT

Rat Hprt1, Cas12a Positive Control crRNA: ACCGCCCCCCCCATACCCCAA

PCR primers

Our scientists have also designed and tested PCR primers (Alt-R HPRT PCR Primer Mixes for human, mouse, or rat) for use
with the Alt-R Genome Editing Detection Kit to detect editing or estimate editing efficiency in samples transfected
with the positive control HPRT crRNAs.

Figure 4. Sample data from T7 endonuclease I (T7EI) assays of samples transfected with Cas12a HPRT Positive Controls. Genomic DNA from CRISPR-Cas12a edited human, mouse, and rat HPRT controls were PCR amplified, digested using T7EI, and run on the Fragment Analyzer™ system (Advanced Analytical Technologies, Inc.). Reference standard bands at 5000 bp (upper marker) and 35 bp (lower marker) are used to align the gel and analyze the results. Estimated band sizes for the cut and uncut positive control amplicons are listed in the table. Cell lines used were HEK-293 (human), Hepa1-6 (mouse), and RG2 (rat). PCR annealing temperatures for human and mouse primers is 67°C and for rat primers is 64°C.

Negative controls

Negative control crRNAs are important for showing that transfection of the RNP complex is not responsible for observed phenotypes.
Amplification of DNA from these negative control samples with your experimental primers and cycling conditions should
result in only full-length products with the Alt-R Genome Editing Detection Kit (i.e., in a T7EI assay). Note, that this
result does not rule out off-target effects of your experimental crRNA.

Our scientists have computationally designed and tested negative control crRNAs to be non-targeting in human, mouse, and
rat genomes. To order, copy and paste the appropriate sequence in the Cas12a (Cpf1) crRNA ordering page:

Cas12a Negative Control crRNA #1: CGTTAATCGCGTATAATACGG

Cas12a Negative Control crRNA #2: CATATTGCGCGTATAGTCGCG

Cas12a Negative Control crRNA #3: GGCGCGTATAGTCGCGCGTAT

Comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1)

Cas9 system

Cas12a system

Applications

General genome editing

For species with AT-rich genomes

For regions with limiting design space for use of the CRISPR-Cas9 system

PAM sites for Cas12a include TTTA, TTTC, and TTTG

A.s. Cas12a nuclease has a lower rate of cleavage for its PAM sequences than S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree. Our scientists have found that crRNAs that have TTTA, TTTC, and TTTG PAM sequences tend to be more functional than crRNAs that have a TTTT PAM sequence (Figure 1).

The electroporation enhancer is required for efficient genome editing with the CRISPR-Cas12a system

Our research shows that not all Cas12a PAM sequences are active sites for genome editing. We recommend that you test 3 or more PAM sites in your region of interest and include the Alt-R Cas12a (Cpf1) Electroporation Enhancer to achieve efficient genome editing (Figure 2). The electroporation enhancer is a non-targeting carrier DNA that shows no integration into the target site based on next generation sequencing experiments.

User guides and protocols

Improved enzymes: The Alt-R Cas12a (Cpf1) enzyme has recently been further optimized to deliver even higher performance. The latest version (V3) can be directly substituted into this protocol in place of the prior Alt-R Cpf1 enzyme.